Part 6, Inactivation Methods Grouped by Virus - 2nd

Part 6, Inactivation Methods Dry heat treatments also produced variable
Grouped by Virus inactivation. Not surprisingly, inactivation
increased with increasing temperature, but the
This article was first published in BioPharm International’s April 2003 issue. effect of moisture can be significant. As noted in
one publication, dry heat (80°C for 72 hours) with
the log reduction values reported are influenced a moisture content у0.8% provided an average
by multiple factors in the viral clearance studies. log10 reduction of 3.72 for PPV, but it was less
Reported log reduction values are limited by the (2.5 log10) with a moisture content р0.8% (11).
starting titer of the virus used in the study, the
virus-to-load spiking ratio, and the sensitivity of Vapor heating has been used to inactivate
the assay used to detect the virus. MMV. A 4.8 log10 inactivation was obtained by
10 hours of 60°C treatment followed by 1 hour
Although the effect of the sample matrix and at 80°C (10).
exact operating ranges must always be
evaluated, these data can provide the reader a The effectiveness of freeze-drying and dry
starting point for assessing inactivation heat treatment (at 80°C for 72 hours) in the
techniques that might be incorporated in a inactivation of CPV and BPV in two different
process or for providing decontamination. factor VIII (FVIII) concentrates was found to be
(Unless specified, ambient inactivation variable and depended on both the virus and the
temperatures were 18°–25°C.) product. The FVIII products were made by two
different facilities. In one FVIII, BPV was more
Gail Sofer, Dorothy C. Lister, Nonenveloped Viruses resistant to inactivation by heat treatment than
and Jeri Ann Boose Single-Stranded DNA viruses CPV. In the second FVIII product, however, there
Diameter: 18–24 nm; Shape: Icosahedral. was no significant difference in the resistance of
In previous articles of this viral inactivation Resistance to physicochemical treatments: Very high. the two parvoviruses. Some factors that were
series (1–6), the virus inactivation methods Parvovirus models: Murine minute virus (MMV), suggested as influences for the variability were
described in the recent literature were sorted by porcine parvovirus (PPV), canine parvovirus ionic composition, differences in the freeze-
test article (bone, red blood cells, and plasma, (CPV), and bovine parvovirus (BPV). drying processes, and moisture content (12).
for example). In this final article, the data from
the literature and from the extensive BioReliance Although these parvoviruses (MMV, PPV, Freeze-drying and heating at even higher
databases have been sorted by virus to enable us CPV, and BPV) are classified in the same family, temperatures (for example at 100°C for 30 min-
to draw conclusions on effective inactivation there is some variability even within this utes on the final container of FVIII concentrate)
methods for a range of viruses. (Data not classification in susceptibility to inactivation was found to inactivate most viruses to below
referenced to a literature citation came from conditions. For example, a recent publication their detection limits. BPV, however, showed
BioReliance databases). indicates that PPV is less likely than human strong resistance to the dry heat treatment (13).
parvovirus B19 (PV-B19), for which it is often a
We selected parvovirus, reovirus (Reo), and model, to be inactivated during pasteurization of pH. Inactivation of PPV by low pH is
hepatitis A (HAV) as models for the human serum albumin (HSA) (7). Some of the variable. For one test article at BioReliance,
nonenveloped viruses, and murine leukemia more commonly used virus inactivation methods complete inactivation was found within 6 hours
virus (MuLV) and pseudorabies virus (PRV) for for these rather hardy viruses include heat, pH, at pH 2.0. However, for that product, it was
enveloped viruses. These viruses were selected irradiation, and chemical treatments. observed that pH treatments from 2 to 5 were
to encompass both single- and double-stranded ineffective. Complete inactivation by low pH
RNA and DNA viruses and to provide a range of Heat. Heating at 58–60°C for 10 hours was found for another product, but this required
resistance to physicochemical inactivation. generally provides a log10 reduction that ranges a contact time of 6 hours, and a pH of less than
from a low of 1.0 to at most 3.9 for MMV and 2.0. Pepsin and low pH treatment were not very
Articles in the literature often use a variety of PPV. In one case, PPV was not inactivated by effective for parvovirus inactivation.
criteria when performing viral clearance studies treatment at 60°C for 15 hours. Treatment of PPV
and interpreting the data. Log reduction values with higher temperatures (80°–82°C) for 72 hours Alkaline conditions increased inactivation
are often reported as “greater than” (Ͼ), and in appears equally as effective as the 58°–60ºC, with increasing concentrations and contact time,
other cases, they are reported as “greater than or 10-hour treatment. In one case, treatment at 74°C ranging from no inactivation up to 5.8 log10. The
equal to” (у). The latter implies complete for 90 minutes provided a log10 reduction of 5.8 log10 reduction was achieved by also raising
inactivation, yet Ͼ is also used (in the literature) 8.4 — complete inactivation. These data from the temperature to 37°C. NaOH (0.1 M) at 60°C
to designate complete inactivation. Furthermore, BioReliance are consistent with those found in was an effective sanitization system for the inac-
the literature. For example, pasteurization at 60°C tivation of viruses that are found in plasma. Two
for 10 hours provided a log10 reduction of 3.4 for minutes of contact with 0.1 M NaOH at 60°C was
PPV (8). Ten hours is considered a requirement sufficient to inactivate CPV below detection
for PPV inactivation at 60°C (9). After 10 hours, limits in these studies (14). Another study,
3.2 log10 of MMV were inactivated (10). however, found that at 60°C, 0.25 M NaOH
inactivated Ͼ3.5 log10 of CPV in 30 minutes, but
inactivation was not complete (р6 log10) (15).

Irradiation. UV irradiation is very effective
for parvoviruses. In one case, it provided a

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Part 6: Inactivation Methods Grouped by Virus

5.5 log10 reduction of PPV, which was complete Resistance to physicochemical treatments: Medium. from two to three hours provided reduction values
inactivation. BPV in fetal calf serum (FCS) was Virus model: Reovirus (Reo). ranging from a low of 2.6 up to 3.6 log10.
completely inactivated by UV irradiation (16).
According to information from the American Acetonitrile was an effective agent of inactiva-
UVC completely inactivated PPV (17,18). Type Culture Collection (ATCC), reoviruses are tion for Reo. Other effective inactivating
UVC also inactivated of both BPV and PPV with ether-resistant and acid- and heat-stable (26). chemicals for this hardy virus include guanidine
log10 reduction factors of 8 for BPV and 5.5 for However, some heating conditions are sufficient hydrochloride, ethanol combined with acetic acid,
PPV (19). Lyophilization combined with gamma for inactivation. Reoviruses are members of the methanol, and bleach. A polyvinylpyrrolidone
irradiation has provided a significant log10 Reoviridae family, which includes blue tongue (PVPP) and iodine complex in a depth filter was
reduction of parvoviruses. In one publication, virus (BTV) that is sometimes used as a model used for viral inactivation of Reo-3 in a 2% IgG
gamma irradiation was reported to inactivate virus. BTV, however, is more sensitive to inac- solution. The filtration speed was 100–500 L
4 log10. The amount of gamma irradiation ranged tivation than reoviruses. BTV is said to be sen- 1/h*2, and the temperature was either 40°C or
from 23 kGy to 30 kGy, depending on the sitive to acid pH, detergents, and lipid solvents. room temperature. Reo was inactivated by
presence of specific plasma proteins (20). 4.9 log10 (the available starting titer) (30).
Heat. Heat treatments of 60°C or higher were
In another article, it was observed that a effective in inactivating Reo. Freeze-drying and Single-Stranded RNA viruses
6 log10 reduction of PPV could be obtained by heating at 100°C for 30 minutes on the final Diameter: 22–30 nm; Shape: Icosahedral.
gamma radiation. The amount of gamma container of FVIII concentrate was found to Resistance to physicochemical treatments: High.
radiation required can vary from 30 kGy to inactivate Reo-3 by 6 log10 (13). Virus model: Hepatitis A (HAV).
100 kGy, depending on the laboratory where the
work was performed and the starting titer (21). At pH. Low pH was not very effective for HAV is a picornavirus; rhinoviruses, which
lower radiation doses, however, PPV may not be inactivating Reo. At pH 2–4, there were only a are more sensitive to acid, are also included in
inactivated. One publication reported that in the few cases in which any inactivation was seen in this family. Most of the picornaviruses inhabit
range of 20–25 kGy, PPV was not inactivated in the BioReliance database. At a pH lower than intestinal tracts, and not all picornaviruses will
FCS (16). More recently, a dose of 34 kGy was 2.0 for one hour, some inactivation was seen for grow in tissue cultures. In addition to HAV, other
found to be necessary for reducing infectivity by two test articles, but in one case a pH of 1.0 was members of the family include encephalo-
4 log10 for BPV in frozen suspensions ineffective even after 24 hours. Pepsin treatment myocarditis (EMC), bovine enterovirus (BEV),
representative of the process used for preparation at pH 4.4 provided some inactivation, but the coxsackieviruses, echoviruses, and polioviruses.
of human cortical bone transplants (22). log10 reduction values were, at most, 2.3. Adding
acetonitrile and lowering the pH enhanced the Heat treatment in solution at temperatures
Electron-beam irradiation has been evaluated inactivation. Alkaline conditions were much from 58°C to 100°C effectively inactivated
for inactivation of PPV and provided log10 more effective than low pH treatments and HAV, from a low of Ͼ2.7 up to 6.5 log10.
reduction of 4.6 in one case. It is reported that provided reduction values up to 7.0 log10. Inactivation times ranged from zero to 16 hours.
extrapolated data indicate a 6 log10 reduction of HAV appears to be more heat-stable than
PPV with a dose of 35 kGy for liquid samples or Irradiation. UV and gamma irradiation were polioviruses and rhinoviruses. The inactivation
55 kGy for frozen samples (23). found to be fairly effective at inactivating Reo. kinetics for HAV during pasteurization of a 5%
Reo in FCS was reduced by about 4 log10 by albumin solution were compared to those of two
Chemical treatments. Ethyleneimines have UV irradiation (16). UVC (220–280 nm) also other picornaviruses. Approximately 7 log10 of
been effective for inactivation of PPV. A 4 log10 provided a 4 log10 reduction (27). Reo in FCS polio type-1 were inactivated in 20 minutes, but
inactivation of PPV took nine hours in a 5-mM was inactivated, but not very quickly, by after 60°C for 10 hours, only 4.8 log10 of HAV
ethylenimine solution and three hours for a gamma radiation in the range of 20–25 kGy were inactivated (10). HAV was efficiently
10-mM solution, both at 37°C (23). Binary (16). Methylene blue photodynamic treatment inactivated in FVIII; however, in a buffer
ethyleneimine (BEI) can also inactivate PPV. A inactivated 3.80 log10 of Reo-3 (28). solution, it was more stable than polio (31).
temperature of 37°C is commonly used (24).
Chemical treatments. Hydrogen peroxide at Protein concentration and stabilizers can also
Inactine (PEN 110) is an electrophilic com- elevated temperatures (50–55°C) provided a affect heat treatment in solution. Inactivation of
pound (positively charged below pH 10), with log10 reduction of Ͼ4. In the literature, HAV by pasteurization in one albumin
low molecular weight, from a family of ethylene- however, avian Reo susceptibility to production process was evaluated and found to
imine oligomer substances. It reacts with nucleic microaerosolized hydrogen peroxide (5%) mist be 4.4 log10 for a 5% albumin solution and
acids to permanently modify the virus genome, varied with the method of exposure. A 10% Ͼ3.9 log10 in a 20% albumin solution (32).
which prevents virus replication and renders the mist was more effective (29). Inactivation of HAV by pasteurization (60°C
virus noninfectious. Inactine provided a Ͼ5 log10 for 10 hours) was investigated in a FVIII
reduction of PPV in human red blood cell con- Formaldehyde provided minimal inactivation product. HAV was substantially inactivated, but
centrates (25). Iodine treatments have also been of Reo in our tests. In most cases, inactivation the stabilizers used in the manufacturing
effective in inactivating PPV, and Iodine/ Sepha- was very low or nonexistent. One exception process delayed the inactivation, and residual
dex (not currently available) has delivered iodine was the treatment with 100 ␮g of formalin per infectious HAV was found even after a 10-hour
to intravenous immunoglobulin in a slow, con- mL at 37°C for three days. heat treatment in the stabilized preparation (33).
trolled way, inactivating Ͼ4.2 log10 in PPV (20).
Urea usually had a significant inactivation A modified pasteurization process (63°C for
Double-Stranded RNA viruses effect, with concentration influencing the inacti- 10 hours) was employed for virus inactivation
Diameter: 60–80 nm; Shape: Spherical. vation. At 4°C and pH 7, 1 M urea was totally of process intermediates of a FVIII plasma
ineffective, but urea concentrations of 3–4 M in a product. Polio, coxsackievirus, and HAV were
S-38 BioPharm International JUNE 2003 Supplement pH range of 4.5–8 with exposure times ranging

inactivated. Virus reduction factors (log10) for 385 mbar. The most intense protocol was for ammonium chloride (DDAC), a quaternary
HAV were у5.6, for polio-1 у9.8, and for fibrinogen, and it employed a pressure of ammonium compound, and 0.1 N NaOH was
coxsackievirus-B6 у4.7. The temperature of 200 mbar and a three-hour treatment at 80°C with evaluated for sanitization studies (15).
63°C was selected rather than 60°C because a pressure of 385 mbar. One-step vapor heating
earlier reports in the literature stated that procedures had the capacity to inactivate between HAV and polio-2 were inactivated by a
temperatures greater than 60°C were required to 5.9 and Ͼ6.3 log10. The two-step procedure hydrogen peroxide gas plasma sterilization
inactivate picornaviruses (34). Heat treatment at inactivated between Ͼ8.7 and 10.4 log10 (39). process. The test viruses were suspended in cell
63°C for 10 hours was also effective for virus culture medium and dried on the bottom of
inactivation in a high-purity FVIII concentrate pH. The effect of pH on the effectiveness of sterile glass petri dishes (44).
stabilized by the von Willebrand factor. In- pasteurization for inactivating HAV was found
process, production samples were stabilized to be variable in fraction V–like products (5% Enveloped Viruses
using amino acids and sugars before spiking. In human plasma protein solution). Increasing the Single-stranded RNA viruses
this case, polio, coxsackievirus, and HAV were pH from 6.4 or 6.5 to 7.5 increased the HAV Diameter: 80–110 nm; Shape: Spherical.
inactivated by Ͼ4.5 log10 (35). inactivation. But in a 5% albumin solution, Resistance to physicochemical treatments: Low.
inactivation was more effective at pH 6.4 than Retrovirus models: Murine leukemia virus (MuLV)
Dry heat appears to be an effective inactiva- 6.9 or 7.4. We concluded that predicting how and xenotropic murine leukemia virus (XmuLV).
tion method; however, in one case, no inactiva- variations in process parameters will affect viral
tion was found at 100°C for one hour. Two high- clearance is difficult and that results will vary Within the retrovirus family are the avian
purity coagulation factors (FVIII and FIX), depending on the virus and the product (40). type C retroviruses, foamy viruses, the human
subjected to dry heat treatment for 24 hours at T-lymphocyte and bovine leukemia viruses
80°C, reduced HAV infectivity was reduced by Sodium hydroxide at concentrations of 0.01 N (HTLV–BLV), lentiviruses, mammalian type-B
у4.3 log10. That reduction was also achieved to 1.0 N has provided a maximum inactivation of oncoviruses, and the viruses related to murine
after two hours and before six hours at 90°C. Ͼ2.7 log10 at low temperatures. It was reported leukemia viruses (MLV). The type-C MuLV —
HAV was also inactivated during the freeze- that treatment of HAV with up to 1.0 M NaOH at the most commonly used model virus — is
drying process by approximately 2 log10 (36). 15°C did not lead to rapid inactivation. However, particularly susceptible to inactivation.
a two-minute contact time with 0.1 M NaOH at
A study of 26 hemophilia patients receiving 60°C was sufficient to inactivate HAV (14). At Heat. At 56–100°C in solution, inactivation
FVIII and FIX products that had been heat treated 60°C, 0.25 M NaOH inactivated Ͼ3.5 log10 of was generally very effective. In one case,
at 100°C for 30 minutes following lyophilization HAV in 30 minutes, but inactivation was however, stabilizers were required. Without the
indicated that the dry-heat treatment is sufficient incomplete (р6 log10). Only 2.7 log10 were stabilizers, there was no inactivation at 60°C.
to inactivate HAV (37). Freeze-drying and inactivated after exposure to 0.1 M NaOH at Except for this unusual case, log10 reduction
heating at 100°C for 30 minutes on the final 25°C for two hours. A 2-hour exposure to 0.5 M values ranged from 3.6–9.2.
container FVIII concentrate was found to NaOH at 4°C reduced HAV only 2.4 log10 (15).
inactivate HAV to below the detection limit pH. At pH values below 3.0, inactivation was
(Ͼ5.3 log10) after a few minutes (13). Demineralization (decalcification) of bone in usually instantaneous; however, the log10 was as
HCl has been shown to inactivate HAV (41). low as 4.1 and as great as 7.1. Acetic acid (1 M)
Heat treatment at 100°C for 30 minutes was inactivated Ͼ4 log10 at either 2°C to 10°C or
sufficient to inactivate HAV and polio-1 by Irradiation. Electron beam, UV, and UVC 18°C. At a pH of 3.5, the lowest log10 reduction
approximately 5 log10 after four minutes in a have been shown to be effective techniques for observed was 3.5. At pH 3.7, instantaneous
FVIII concentrate (38). Moisture content influ- inactivating HAV. A fibrinogen solution inactivation was sometimes obtained, but for one
ences the effectiveness of dry heat (80°C, containing rutin to protect the fibrinogen was test article the reduction was only 1 log10. In
72 hours) for viral inactivation in lyophilized irradiated with UVC (254 nm at 0.1 J per cm2). another study on pH 3.7 inactivation, however,
FVIII. When the moisture content was у0.8%, HAV was inactivated by у6.5 log10 (17). UVC the log10 reduction was 5.6. At pH 3.8, log10
the average log10 reduction for HAV was у4.54; irradiation was also used for virus inactivation reduction values were as high as 7, but in two
when moisture content was р0.8%, however, the in plasma and in a FVIII concentrate. HAV in cases only a 1.7 log10 reduction was found.
average log10 reduction was 0.12 (11). the FVIII concentrate and in the plasma was
inactivated to undetectable levels (у6.3) (18). One study presented at a conference
Vapor heating (lyophilization, followed by described pH 3.5–4.0 to be effective at 18°C to
adding water to achieve homogenous moistening, The antigenicity of HAV was shown to be 26°C, and very little difference was seen in
then heating at 60°C for 10 hours, followed by almost unaltered after UV treatment in a study to inactivation kinetics between pH 3.7 up to
heating at 80°C for one hour in an airtight determine the suitability of UV inactivation for 4.1. At 2°C to 8°C, however, pH 4.1 inactivation
container under nitrogen gas atmosphere) of a vaccine preparation. UV doses up to 920 J/m2 was slower and required up to an hour; only
FVII concentrate provided HAV inactivation were applied. UV treatment preserved antigeni- about 30 minutes were needed for pH 3.7 (45).
within one hour at 60°C (5.9 log10) (10). Vapor city at levels comparable to treatment with
heating was evaluated for its capacity to inacti- formalin at 250 ␮g/mL, but formalin requires up At pH 4, log10 reduction values ranged from
vate HAV in six different coagulation factor to 15 days to achieve inactivation (42). 3.2 to 4.9. At pH 4.5 for 45 minutes, log10
concentrates, fibrin sealant, lys-plasminogen, and reduction values ranged from a low of 1.1 up to
fibrinogen. Vapor heating conditions were vari- Chemical treatments. Iodine at 37°C reduced 4.7. In one case, however, two hours at pH 4.5
able and depended on the stability of the different HAV by 3.8 log10 after six hours. An HAV produced no measurable inactivation. A
products, but all included a 10-hour, 60°C treat- vaccine treated with 0.2 mg/mL formaldehyde 16-hour cold-room treatment at pH 4.5 provided
ment and pressures ranging from 190 mbar to for five days at 32°C was found to be safe and a 4.8 log10 reduction, and 48 hours at 2–20°C
immunogenic in experimental models (43). gave a log10 reduction greater than 5.9 for one
HAV inactivation with 0.1% didecyldimethyl- test article. At pH values greater than 5,

BioPharm International JUNE 2003 Supplement S-39

Part 6: Inactivation Methods Grouped by Virus

inactivation is typically low. In one case with Tweens, even at low temperatures, and Triton compensate for low temperatures. For example, at
low conductivity, a log10 reduction of X-100 was also highly effective in most cases. pH 4.0, 2°C to 10°C, a log10 reduction of
approximately 3 was obtained at a pH of 6.9. Ͼ5.7 was achieved for a two-day inactivation
In one study, it was shown that Triton X-100 treatment. At room temperature, the log10
In addition to the influence of time and (0.5% at 4°C) completely inactivated Friend reduction was Ͼ5.3 after a 20-minute treatment.
temperature on pH inactivation, it has been murine leukemia virus (Fr-MuLV) within four In another case, it did not appear to be necessary
observed that different products and different hours without influencing the binding capacity to extend the time when the temperature was
protein concentrations affect the ability of low of a number of monoclonal antibodies. Log10 lowered. At pH 3.5–3.7, a 2–8°C temperature
pH treatments to inactivate MuLV. Abujoub reduction values were у3.8 for Fr-MuLV (46). produced a Ͼ5.7 log10 reduction within one hour.
described an inactivation time for one product At temperatures of 15°C and higher, log10
of 60 minutes and 120 minutes for another (45). A review of viral safety discusses the success- reductions of Ͼ6.4 were achieved in one hour.
Furthermore, in one product, X-MuLV was not ful use of S/D. S/D has been shown to completely
completely inactivated even after 120 minutes. inactivate MuLV (у6.0) (47). Complete When pH was raised to 5, log reduction
Protein concentration also affected the inactivation was also reported for a recombinant values were severely reduced: In most cases, no
inactivation kinetics. In buffer only, X-MuLV blood factor (48). In S/D inactivation of MuLV, reduction was found. Even with the addition of
was inactivated in 120 minutes. The addition of Tween from vegetables has been found to be as phenol and a one-day treatment, there was no
protein prevented complete inactivation with effective as Tween derived from animals. inactivation of PRV at pH 5.5.
the same pH, temperature, and exposure time.
Double-Stranded DNA viruses Low pH was investigated for inactivation of
Under alkaline conditions, log10 reduction Diameter: 120–2-0 nm; Shape: Spherical. PRV in several monoclonal antibodies produced
values ranged from 4.1 to Ͼ6.2 at NaOH Resistance to physicochemical treatments: Medium. in either Sp2/0 or NS0 cell lines. Log10 reduction
concentrations ranging from 0.01–1 N. Tempera- Virus model: Herpes group, pseudorabies virus. ranged from a low of 3.78 to complete inactiva-
ture influenced the effectiveness of the inactiva- tion at pH ranging from 3.32 to 3.51 (50).
tion. Not surprisingly, at lower temperatures, The herpes group of viruses includes herpes
inactivation by alkaline conditions was reduced. simplex (HSV), bovine rhinotracheitis virus Virus inactivation with pH 4/pepsin has been
(BRV), cytomegaloviruses (CMVs), and used as part of the manufacturing process for
Irradiation. Electron beam treatment gave a varicella-zoster virus (VZV). PRV is commonly intravenous IgG. Factors found to influence the
log10 reduction of 1.3–1.9, and gamma irradiation used as the model for herpes virus. inactivation kinetics include temperature and
produced a log10 reduction of Ͼ5.5. Methods Heat. Rabbit serum heated 30 minutes at 56°C content of the IgG solution. At 37°C, complete
such as electron beam and gamma irradiation are provided fairly effective inactivation for PRV, but inactivation of PRV required 30 minutes. Below
being applied to new technologies such as tissue the researchers found that PRV is usually resis- 37°C, there was a marked decrease in virus
therapies, and the database of inactivation tant to heat in the presence of protein stabilizers inactivation with the lowest rates at 4°C. At
information is expected to expand in this area. (16). Heating in solution at temperatures from neutral pH, where no pepsin activity was
60°C to 80°C can, however, often effectively detectable, virus inactivation was negligible at
Chemical treatments. Ethanol at 20% provided inactivate PRV by 4.1 to 8.1 log10. Inactivation both 37°C and 4°C. Increasing sucrose
no inactivation, but concentrations of ethanol kinetics of pasteurization (60°C) were studied for concentrations in the IgG solutions inhibited the
ranging from 30–70% always gave a log10 an antithrombin III product, and it was found that rate of PRV inactivation of PRV. Increases in
reduction Ͼ4. Inactivation by methanol and 30 minutes were required to inactivate 6.6 log10 IgG concentrations had no clear effect on PRV
hexane was not as effective. Log10 reduction of PRV (10). A modified pasteurization process inactivation (51).
values ranged from Ͻ1–2.5. Trifluoroacetic acid (63°C for 10 hours) of process intermediates for a
(TFA) at a low pH gave good inactivation, but at FVIII plasma product provided viral inactivation Alkaline conditions were effective at inacti-
a neutral pH or at lower temperatures (2–8°C), of PRV at у5.3 log10 (34). vating PRV. At a NaOH concentration as low as
there was no inactivation. Acetonitrile was 0.1 N, inactivation was effected within a few
effective in all cases, providing a 3.8 to Dry heat at temperatures of 59°C to 100°C hours. Inactivation by 0.5 N NaOH for two-hours
Ͼ5.1 log10 reduction at times ranging from zero does not appear to be as effective as heating in was effective at temperatures as low as 4°C. One
to four hours. Bleach was also found to be solution. Inactivation times for heat treatments study achieved PRV inactivation by a two-minute
effective. A 4.5 log10 reduction was observed at ranged from zero to 72 hours. Lyophilization contact with 0.1 M NaOH at 60°C (14).
time zero with 600 ppm hypochlorite. without heat treatment provided no inactivation in
some cases and Ͼ4.4 log10 reduction in one case. Irradiation. Gamma irradiation was highly
Urea is an effective inactivating agent for effective, providing Ͼ5.6 log10 reduction. PRV
MuLV, either with or without acetonitrile. A minimum moisture content у0.7% is inactivation by gamma irradiation has also been
Concentrations of 2–6 M urea were used. necessary for virus reduction in the magnitude of described by Pruss et al. (22). Light and mero-
Guanidine hydrochloride (4 M at pH 7) 4 log10 for PRV in lyophilized factor VIII (11). cyanine 540 inactivated PRV by 6 log10 in the
provided a 3.8 log10 reduction at time zero. Freeze-drying and heating at 100°C for absence of added protein. But when 16% plasma
30 minutes on a final container FVIII concentrate was added, reduction was only 1.3 log10 (52).
Detergents alone may not be sufficient to inactivated PRV by 5.7 log10 (13). Vapor heating
inactivate lipid-enveloped viruses such as of a FVII concentrate inactivated 6.9 log10 PRV Methylene blue (MB) and dimethylmethyl-
MuLV. In some cases, we have observed that in three hours (10). Another article describes a ene blue (DMMB) activated by light at a
MuLV was not sufficiently inactivated by 0.1% Ͼ11.5 log10 for PRV by vapor heating of a wavelength of 630 nm inactivated PRV by
to 1% Tween: Sometimes, there was no coagulation factor concentrate (49). Ͼ5.0 log10 (53).
inactivation at all. From a review of the data,
Tween 20 appears to be more effective than other pH. Low pH (1.7–4.0) inactivation was Chemical treatments. Ethanol alone or
effective. Increasing the time of inactivation can combined with acetic acid was effective for
S-40 BioPharm International JUNE 2003 Supplement inactivation of PRV, as were methanol and

guanidine hydrochloride. Urea was effective with variables were sought that affect viral (8) Chang, C.E. et al., “Human Intravenous
one exception: At a temperature of 2–8°C, urea inactivation. These findings are not unique nor Immunoglobulin Preparation and Virus
provided only a 1.4 log10 reduction after one hour are they all-encompassing. Reviews published Inactivation by Pasteurization and Solvent
of exposure. Ether at low temperatures had only a while these data were being prepared include Detergent Treatment,” Prep. Biochem.
small effect (a 1.4 log10 reduction). Acetonitrile at one that addresses blood components (64), one Biotechnol. 30(3), 177–197 (2000).
a 10% concentration and a pH of 7.8 had no that addresses plasma derivatives (65), and one
effect, but 50% acetonitrile inactivated prepared by the Council of Europe Expert (9) Coan, M.H. et al., “Characterisation and Virus
Ͼ4.7 log10 in four hours. Iodine, hydrogen Committee in Blood Transfusion that addresses Safety of Alpha 1-Proteinase Inhibitor,” Eur.
peroxide, detergents, and solvent/detergent labile blood products (66). Examining the data Respir. J. Suppl., 35s–38s (9 March 1990).
treatments were all effective in our studies. from publications and from our databases
reinforces the opinion that the nature of both the (10) Barrett, P.N., “Determination of the Inactivation
A ␤-propiolactone concentration of 0.1% inactivation process and the product can Kinetics of Hepatitis A Virus in Human Plasma
(w/w) was used for virus inactivation in a 4% strongly influence viral inactivation. Products Using a Simple TCID50 Assay,” J. Med.
immunoglobulin solution. At 22°C, PRV was Virol. 49(1), 1–6 (1996).
inactivated below detection after 80 minutes (54). Many variables affecting viral inactivation are
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